Methylenetetrahydrofolate reductase (MTHFR) is a flavoprotein catalyzing the reduction of 5,10-methylenetetrahydrofolate (CH2-H4folate) to 5-methyltetrahydrofolate (CH3-H4folate). The active form of E. coli MTHFR is a homotetramer which contains both symmetrical and asymmetrical interfaces. In this project, we used hydrogen deuterium exchange monitored by mass spectrometry (HDX MS) to first look at the effect of concentration on the dissociation of MTHFR; second, we explored the dynamics of MTHFR by comparing HDX of different mutants. The mutants of interest are Glu28Gln (E28Q), Glu28Asp (E28D) and Ser26Ala (S26A). By comparing the deuteration levels across these mutants with wild type enzyme, we found out that all these mutants have higher relative deuteration levels compare to wild type which suggested that the mutations in these sites cause the overall loosening of MTHFR. The concentration study compared the deuteration levels at three different concentrations (15, 0.5 and 0.25 µM) and showed that there was no difference in relative deuteration between 0.5 and 15 µM and a slight increase for the deuterium uptake at 0.25 µM. At the six-hour deuteration time point, both 0.5 µM and 0.25 µM exhibited slightly higher deuterium levels than 15 µM MTHFR. Our results imply that there is no dissociation because of change in concentrations, though it is possible that the technique we use is not sensitive to dissociation.

Effect of concentration and mutation on the conformational dynamics of MTHFR

2019 Spring National Meeting

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